Isoniazid (INH) is a frontline drug in tuberculosis treatment that is both potent and selective, yet 50 years after its discovery, its mechanisms of action are not completely defined. INH is a prod rug that is activated by the mycobacterial peroxidase KatG to produce a range of reactive free radicals that react with various mycobacterial components to cause therapeutic effect. It is also known that Mycobacterium tuberculosis is exceptionally sensitive to nitric oxide (NO-) a free radical known to be important in the bodies immune response against TB. The applicant will test the innovative hypothesis that the free radical nitric oxide is formed by KatG oxidation of INH, and plays an important role in the overall action of INH. Thus, the exquisite sensitivity of M. tuberculosis to INH may be, in part, the result of its extreme sensitivity to NO- formed by INH activation. Initial data generated by the applicant does show that KatG oxidation of INH generates NO-, that an NO- scavenger can protect mycobacteria against INH, and that mycobacterial enzymes known sensitive to NO- are inhibited by INH. However, for these findings to be widely accepted, and before in-depth studies are conducted, key details on the mechanism of NO- production, and its overall importance in INH action must be addressed. Specific Aim 1 will characterize the pathway of NO- formation by KatG activation of INH, EPR spin trapping, and NMR product studies in combination with specific stable isotopic labeling of INH. The S315T KatG mutation associated with INH resistance will be studied to determine if lowered NO production may contribute to resistance in this mutant. Specific Aim 2 will examine the contribution of NO- to the overall action of INH by using a range of specific NO- scavengers to determine the maximal protection that optimized NO scavenging provides M. tuberculosis against INH. We will also use RT-PCR and enzymic approaches to compare mycobacterial defense responses signatures to NO- and INH and determine the extent to which the mycobacterial response to INH is related to its known response to NO-. [unreadable] [unreadable] [unreadable]